The use of optical fibers and optical fiber strands in combination with light energy absorbing dyes for medical, biochemical, and chemical analytical determinations has undergone rapid development, particularly within the last decade. The use of optical fibers for such purposes and techniques is described by Milanovich et al. "Novel Optical Fiber Techniques for Medical Application", Proceedings of the SPIE 28th Annual International Technical Symposium on Optics and Electro-Optics, Volume 494, 1980; Seitz, W. R., "Chemical Sensors Based on Immobilized Indicators and Fiber Optics" in C.R.C. Critical Reviews In Analytical Chemistry, Vol. 19, 1988, pp. 135-173; Wolfbeis, O. S., "Fiber Optical Fluoresensors in Analytical Chemistry" in Molecular Luminescence Spectroscopy, Methods and Applications (S. G. Schulman, editor), Wiley & Sons, New York; Angel, S. M., Spectroscopy 2(4): 38 (1987); and Walt et al., "Chemical Sensors and Microinstrumentation", ACS Symposium Series, Volume 403, 1989, p. 252.
The optical fiber strands typically are glass or plastic extended rods having a small cross-sectional diameter. When light energy is projected into one end of the fiber strand (conventionally termed the "proximal end"), the angles at which the various light energy rays strike the surface are greater than the critical angle; and such rays are "piped" through the strand's length by successive internal reflections and eventually emerge from the opposite end of the strand (conventionally termed the "distal end"). Typically bundles of these strands are used collectively as optical fibers in a variety of different applications.
For making an optical fiber into a sensor, one or more light energy absorbing dyes are attached to the distal end of the optical fiber. The sensor can then be used for both in-vitro and/or in-vivo applications. As used herein, light energy is photoenergy and is defined as electromagnetic radiation of any wavelength. Accordingly, terms "light energy" and "photoenergy" include infrared, visible, and ultraviolet wavelengths conventionally employed in most optical instruments and apparatus; the term also includes the other spectral region of x-ray and microwave wavelengths (although these are generally not used in conjunction with optical fibers).
Typically, light from an appropriate energy source is used to illuminate what is chosen to be the proximal end of an optical fiber or a fiber bundle. The light propagates along the length of the optical fiber; and a portion of this propagated light energy exits the distal end of the optical fiber and is absorbed by one or more energy absorbing dyes. As conventionally used, the light energy absorbing dye may or may not be immobilized; may or may not be directly attached to the optical fiber itself; may or may not be suspended in a fluid sample containing one or more analytes of interest to be detected; and may or may not be retainable for subsequent use in a second optical determination.
Once the light energy has been absorbed by the dye, some light energy of varying wavelength and/or intensity returns through the distal end of the optical fiber and is conveyed through either the same fiber or a collection fiber or fibers to a detection system where the emerging light energy is observed and measured. The interactions between the light energy conveyed by the optical fiber and the properties of the light absorbing dye--in the presence of a fluid sample containing one or more ligands or analytes of interest and in the absence of any ligands or analytes whatsoever--provide an optical basis for both qualitative and quantitative determinations. Merely illustrating the use of optical fiber sensors presently known in a variety of conditions, apparatus, dyes and applications are U.S. Pat. No. 4,822,746; 4,144,452; 4,495,293; and Re. 31,879.
Most light detection systems employ a photosensitive detector such as a photodiode or photomultiplier tube. Spatial resolution of light is possible with two dimensional detectors such as video cameras and charge coupled devices. Moreover, in view of the microcircuitry and enhanced television technology presently available, a variety of light image processing and analytical systems have come into existence in order to both enhance, analyze and mathematically process the light energies introduced to and emerging from the absorbing dyes in such optical analytical techniques. Typically, these systems provide components for photon measurement and include image capture; data acquisitions; data processing and analysis; and visual presentation to the user. Commercial systems available today include the QX-7 image processing and analysis system sold by Quantex, Inc. (Sunnydale, CA); and the IM Spectrofluorescence imaging system offered by SPEX Industries, Inc. (Edison, N. J.). Each of these systems may be combined with microscopes, cameras, and/or television monitors for automatic processing of all light energy determinations.
Of the many different classes of light absorbing dyes which conventionally are employed with bundles of fiber strands and optical fibers for different analytical purposes are those compositions which emit light energy after absorption, termed "fluorophores"; and those which absorb light energy and internally convert the absorbed light energy rather than emit it as light, termed "chromophores." Fluorophores and fluorescent detection methods employing optical fibers are recognized as being markedly different and distinguishable from light energy absorbance and absorption spectroscopy.
Fluorescence is a physical phenomenon based upon the ability of some molecules to absorb light energy (photons) at specified wavelengths and then emit light energy of a longer wavelength and at a lower energy. Such emissions are called fluorescence if the emission is relatively long-lived, typically 10.sup.-11 to 10.sup.-7 seconds. Substances able to fluoresce share and display a number of common characteristics: the ability to absorb light energy at one wavelength or frequency; the reaching of an excited energy state; and the subsequent emission of light at another light wavelength and energy level. The absorption and fluorescence emission spectra are thus individual for each fluorophore; and are often graphically represented as two separate curves which are slightly overlapping. Also, all fluorophores demonstrate the Stokes' Shift--that is, the emitted light is always at a longer wavelength (and at a lower energy level) relative to the wavelength (and energy level) of the exciting light absorbed by the substance. Moreover, the same fluorescence emission spectrum is generally observed irrespective of the wavelength of the exciting light and, accordingly, the wavelength and energy of the exciting light may be varied within limits. Finally, fluorescence may be measured as the quantum yield of light emitted. The fluorescence quantum yield is the ratio of the number of photons emitted in comparison to the number of photons initially absorbed by the fluorophore. For more detailed information regarding each of these characteristics the following references are recommended: Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Plenum Press, New York 1983; Freifelder, D., Physical Biochemistry, second edition, W. H. Freeman and Company, New York, 1982; "Molecular Luminescence Spectroscopy Methods and Applications: Part I" (S. G. Schulman, editor) in Chemical Analysis, Vol. 77, Wiley & Sons, Inc., 1985; The Theory of Luminescence, Stepanov and Gribkovskii, Iliffe Books, Ltd., London, 1968.
In comparison, substances which absorb light energy and do not fluoresce usually convert the light energy into heat or kinetic energy. The ability to internally convert the absorbed light energy identifies the dye as a "chromophore." Dyes which absorb light energy as chromophores do so at individual wavelengths of energy and are characterized by a distinctive molar absorption coefficient at that wavelength. Chemical analyses employing fiber optic strands and absorption spectroscopy using visible and ultraviolet light wavelengths in combination with the absorption coefficient allow for the determination of concentration for specific analytes of interest by spectral measurement.
Many of the recent improvements employing optical fiber sensors in both qualitative and quantitative analytical determinations concern the desirability of depositing and/or immobilizing various light absorbing dyes at the intended distal end of an optical fiber using a given technique or apparatus. In this manner, a variety of different optical fiber chemical sensors and methods have been reported for specific analytical determinations and applications such as pH measurement, oxygen detection, and carbon dioxide analyses. These developments are exemplified by the following publications: Freeman et al., Anal. Chem. 53:98 (1983); Lippitsch et al., Anal. Chem. Acta. 205:1, (1988); Wolfbeis et al., Anal. Chem. 60:2028 (1988); Jordan et al., Anal. Chem. 59:437 (1987); Lubbers et al., Sens. Actuators (1983); Munkholm et al., Anal. Chem. 58:1427 (1986); Seitz, W. R. Anal. Chem. 56:16A-34A (1984); Peterson et al., Anal. Chem. 52:864 (1980); Saari et al., Anal. Chem. 54:821 (1982); Saari et al., Anal. Chem. 55:667 (1983); Zhujun et al., Anal. Chem. 56:2199 (1984); Collison, M. E. and M. E. Meyerhoff, Anal. Chem. 62:425A (1990); Demas, O. N. and B. A. DeGraff, Anal. Chem. 63:809A (1991); Seitz, W. R., CRC Crit. Rev. Anal. Chem. 19:135 (1988); Kopelman et al., Science 258:778 (1992); Janata, J., Anal. Chem. 64:196R (1992); and Orella et al., Anal. Chem. 64:2210 (1992).
Despite these many innovations and developments, and without regard to whether the application is intended for in-vitro or in-vivo use, it has been nearly impossible to both view the sample or the surrounding environment and concurrently measure and detect a ligand or analyte of interest in a fluid sample using a single optical fiber sensor. Fiber sensors are usually single fibers, thereby precluding their use for imaging. On the other hand, imaging fibers and arrays have been employed only for their intended purpose--viewing. Thus, if viewing is desired, a separate optical fiber coherent bundle which is unencumbered and unobscured would be required in order to view the sample and its surroundings directly. Each of these chemical sensing fibers and visualization fibers markedly increases the size and complexity of the overall system such that no apparatus or system presently exists which both chemically senses and provides viewing capability concurrently. Accordingly, the development of a single imaging fiber optical sensor able to utilize a dye reagent for optical determinations of different ligands of interest, concurrently with a capability of viewing the sample and its environment at will, would be recognized as a major advance and substantial improvement.